Interpretation tools for coreceptor usage Rolf Kaiser Institute of - PowerPoint PPT Presentation

Interpretation tools for coreceptor usage Rolf Kaiser Institute of Virology University of Cologne

Coreceptor Usage � 2 Chemokinreceptors are used in vivo as coreceptors: CCR5 & CXCR4 � CCR5 only (R5-Virus) � CXCR4 only (X4-Virus) � Viruses using CCR5 and CXCR4 (R5/X4-Virus, dualtropic) � Mixtures of R5- and X4-Viruses in patients plasma (Mixed) � For clinical purpose 2 classes: � CXCR4 can be used � CXCR4 cannot be used => R5 viruses that can be inhibited by R5-blockers

R5/X4 Tropic HIV HIV Entry Cell Assay (Trofile) CD4 + CXCR4 + HIV env HIV genomic expression luc vector vector + + + Infection Transfection Pseudovirus - capable of a single round of replication CD4 + CCR5 + CCR5 and CXCR4 antagonists are used to confirm tropism Adapted from Petropoulos CJ, et al. Antimicrob Agents Chemother. 2000;44:920-8.

deCIPhR for Entry inhibitors β -Gal X4 Pr Env T20 X4-3026 SCH417690 TF141-1 AMD3100 TAK779 Av. %Inh. % virus inhibition 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 0 -20 -20 -20 0.1 10 1000 0.01 1 100 0.01 1 100 Cpd. conc. (nM) Cpd. conc. (nM) Cpd. conc. (nM) Fusion Inhibitors CXCR4-antagonists CCR5-antagonists AMD3100 T20 SCH417690 X4-3026 TF141-1 TAK779 Av. %Inh. Av. %Inh. Av. %Inh. 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 0 -20 -20 -20 0.1 10 1000 0.1 10 1000 0.01 1 100 Cpd. conc. (nM) Cpd. conc. (nM) Cpd. conc. (nM) β -Gal R5 Pr Env

HIV-1 PHENOSCRIPT-ENV � EUROFINS-TRT � Data presented from JL Faudon

Comparison of different phenotypic assays Monogram-Trofile R5 X4 Eurofins-TRT n=42 (56,8%) R5 n=3 (4.1%) n=21 (28,4%) X4 n=8 (10%) 14% discordant; different cell lines, other differences? Skrabal et al J. Clin. Microbiol, 2007

Questions � Predicting coreceptor usage by genotypic analyses? � Can we simply adapt our experience from resistance testing of RT and Protease? � What gene(-region) to sequence? � aa position 11 and 25 in env-V3 are predictive enough for R5/X4 (SI/NSI) (Fouchier et al 1995)

6558 TGGGATCAAAGCCTAAAGCCATGTG GGGTTCTTRGGAGCAGCAGGAAGCACT 7785 V3 TAATTGTGGAGGGGAATTTT 7371 6957 GTACAATGTACACATGGAAT Izopet et al. Lehmann et al.

Genotypic Monitoring of Coreceptor Usage � 1110 matched genotype (V3 region)-phenotype pairs: genotyping phenotyping multiple alignment (against fixed reference alignment) CTRPNNNTRRSISIGPGRAFYATGDIIGDIRQAHC R5 CTRPNNNTRKGIHMGPGS-FYVTGEIIGDIRQAHC R5 CSRPNNNTRKSVHIGPGQAFYATGDVIGDIRQAHC X4 X=(X 1 , …, X i , …, X n ) Y statistical learning T.Sing

Internet-Tools for genotype based predictions Web-PSSM (Mark Jensen et al),http://ubik.microbiol.washington.edu/computing/pssm/ PSSMs for Subtyp B vs. C* WetCat: (Pillai et al.)http://genomiac2.ucsd.edu:8080/wetcat/v3.html geno2pheno[coreceptor] http://www.genafor.org

geno2pheno[coreceptor] Current geno2pheno[coreceptor]: Specificity 90%, Sensitivity 76% for X4 trained on 1100 Los Alamos data

Geno2pheno[coreceptor]: Output Alignment relative to HXB2 Predicted coreceptor usage

Question � Other attributes ?

Gp120 and V3 region

geno2pheno[coreceptor] For 90% Spec. for X4 Sensitivities are SVM-Seq: 79.4% SVM-struct. descriptors 84,7% SVM-Seq+ struct. descriptors 86,4%. Poster #66 – O. Sander

Genotyping results • Salvage Patients Uni Köln, longitudinal study over 3 years C.Lehmann, G.Fätkenheuer, M.Däumer, R.Kaiser, Journal of Clinical Virology 37 (2006) 300–304 • Successful Amplification in 45 /45 patients (100%) • Interpretation with geno2pheno in 42/45 25 patients (56%) contiunously displayed an R5-Virus 12 patients (27%) contiunously displayed an X4-Virus In1 patient HIV ‚shifted‘ from R5 to X4 In 2 patients HIV ‚shifted‘ from X4 to R5 In 1 patient HIV ‚shifted‘ from X4 to R5 to X4 In 1 patient HIV ‚shifted‘ from R5 to X4 to R5 • In Motivate Studies 8% switched between screening and start of MVC-Therapy

Genotyping results Presentation in Dublin from the “ANRS” - group: Cathia Soulié, Anne Derache, Catherine Aimé, Anne-Geneviève Marcelin, Guislaine Carcelain, Anne Simon, Christine Katlama, Vincent Calvez • Successful Amplifikation in 83,1% of n=69 patients samples • Interpretation with geno2pheno in 66/69 cases, with PSSM in 58/69 cases (No Alignment Tool in PSSM) „mean of both systems“ ART naive: R5 71,2%, X4 28,8% ART experienced R5 58,8%, X4 41,2% • Comparable numbers with results from phenotyic studies?

Percentage of HIV Co-receptor Usage Study/Source Population N R5 X4 R5/X4 Homer cohort 1 Naive 979 82% <1% 18% C & W cohort 2 Naive 402 81% <1% 19% Demarest 3 Naive 299 88% 0% 12% TORO 1/2 4 Experienced 612 62% 4% 34% ViroLogic 5 Experienced >2000 48% 2% 50% ACTG 5211 6 Experienced 391 49% 4% 47% *This table may not include all available reported data. Majority of data are generated in the developed world (subtype B) 4 Whitcomb JM, et al. CROI 2003. Abstract 557. 1 Brumme ZL, et al. J Infect Dis. 2005;192:466-474. 5 Paxinos EE, et al. ICAAC 2002. Abstract 2040. 2 Moyle GJ, et al. J Infect Dis . 2005;191:866-872. 6 Wilkin T, et al. CROI 2006. Abstract 655. 3 Demarest J, et al. ICAAC 2004. Abstract H-1136.

� Phenotypic and genotypic tests came to comparable numbers for coreceptor usage in therapy naives and therapy-experienced patient cohorts

� Minorities � do they always exist? � Do they always play a clinicilly relevant role?

� Are Coreceptor blockers dangerous and drive coreceptor switch?

MOTIVATE 1 and 2: Change in CD4 Cell Count from Baseline by Tropism Result at Time of Failure Mean change in CD4 count from baseline, cells/mm 3 in patients with treatment failure Placebo + MVC QD + MVC BID + Tropism result, OBT OBT OBT Baseline → Treatment Failure N = 209 N = 414 N = 426 All treatment failures +14 +49 +71 (n = 97) (n = 68) (n = 77) R5 → R5 +15 +61 +138 (n = 80) (n = 18) (n = 17) R5 → D/M or X4 +67 +37 +56 (n = 4) (n = 31) (n = 32) � Approximately 8% of patients had a change in tropism result between screening and baseline, demonstrating the change in background tropism over a 4–6 week period in this population � Response to maraviroc treatment in these patients was consistent with the results of study A4001029 in patients with D/M-tropic virus ( XVI IAC 2006, Abs THLB0215) Data excludes patients who had no tropism result at time MOTIVATE 1 & 2-Week 24 of failure and patients with non-R5 virus at baseline

� Resistance to Coreceptor blockers?

HIV-1 isolate from a Δ 32/wt CCR5 patient with unusual broad coreceptor usage V1 V2 V3 C4 region isolate unusual long region Y308 I317 D321 K442 E444 (compensatory) Gorry et al., Virology, Jan 2007

Mutations under MVC pressure during cell culture passage Passage V1 V2 V3 C3 C4 V4 CTRPNNNTRKSI HI GPGRAFYTTGDI I GDI RQAHC CC1/ 85 st art T163 A316 T319 I323I/ V N335N/ P/ K/ Y S405S/ A/ T/ Esuscept ible number 16 T163K A316T/ S T319T/ S I323V N355Y S405A resistent revert ant A316 I323V suscept ible CTRPNNNTRKSI SFGPGQAI YTTGNI I GDI RQAHC RU570 st art S132 QAI 315- 317 T319 N322 suscept ible Δ QAI number 18 (8) S132N N322D N442K D389N resistent number 18 (2) N322D suscept ible revert ant T319K/ T N322D suscept ible Westby et al., Journal of Virology, Mar. 2007

Clinical data that may improve the prediction

� Comparison of genotypic and or phenotypic data see Posters #53 N. Chueca and #82 E. Poveda � Minorities are there but not always a problem X4 under immune control? � We canot simply transfer our tools from RT- and Protease resistance to coreceptor prediction tools

geno2pheno Institute of Virology, University of Cologne Martin Däumer Saleta Sierra-Aragon Eva Heger Claudia Müller Dörte Hammerschmidt Jens Verheyen Ibrahim Boussaad Eugen Schülter Herbert Pfister Rolf Kaiser MPI for Informatics, Saarbrücken Tobias Sing Achim Buech Andre Altmann Alexander Thielen Oliver Sander Thomas Lengauer Dept. of Mathematics, UC Berkeley, CA, USA Niko Beerenwinkel University of Essen; caesar, Bonn Daniel Hoffmann caesar, Bonn Ulrike Schuldenzucker MPl of Molecular Plant Physiology, Golm Joachim Selbig Institute of Clinical and Molecular Virology, German National Reference Center for Retroviruses, University of Erlangen-Nürnberg Barbara Schmidt Hauke Walter Klaus Korn Dept. of Gastroenterology, University of Düsseldorf Mark Oette Dept. of Internal Medicine I, University of Cologne Gerd Fätkenheuer Clara Lehmann Dept. of Internal Medicine I, University of Bonn Jürgen Rockstroh Medical Laboratory, Berlin Thomas Berg PZB, Aachen Heribert Knechten, Patrick Braun and the Resina study group www.geno2pheno.org / www.genafor.org

� Collaborative Trials for Phenotypic Assays � Collaborative Trials for Genotypic Assays => ENVA?

TG_66 � day 0

TG_66 � day 28

TG_66 � day 56

TG_66 3068 � 3164 � 3015 � •X4-Virus as minor variant at day 0. •Day 0 sample showed a T215D revertant •Primary resisant virus

TG_66